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Researchers were interested in using a reporter gene that could be stably expressed in alphaviruses without attenuating infectivity. cDNA clones of eastern equine encephalitis (EEEV), Venezuelan equine encephalitis (VEEV), Sindbis (SINV) and chikungunya (CHIKV) viruses had either firefly luciferase or a FLAG-tagged NanoLuc™ luciferase genes inserted in the genomes using three different insertion points. The cDNAs were then transcribed to generate infectious viral RNAs that were then electroporated into BHK-21 cells, virus particles harvested from the supernatant after 18–24 hours and stored at –80°C in single-use aliquots as viral stock.

To assess how the reporter genes affected viral replication, BHK-21 cells were infected at a multiplicity of infection (MOI) of 0.1 PFU/cell or 5 PFU/cell. After 1 hour, cells were washed and medium replaced. At time points 0, 6, 12, 18, 24 and 48 hours, supernatant was sampled for plaque assay titration and cells lysed with 1X Passive Lysis Buffer for measuring reporter activity using the Luciferase Assay System for firefly luciferase or Nano-Glo® Luciferase Assay for NanoLuc™ luciferase.

To examine how the presence of the reporter gene might affect viral infectivity over time, BHK-21 cells were infected with SINV reporter viruses at an MOI of 0.1 PFU/cell and passage 1 (P1) viral stock was harvested 18 hours after infection. The SINV virus was then diluted 1:1,000 for infection of fresh cells, serially passaged nine more times. Supernatants from P1– P10 viruses were titrated by plaque assay; cells were lysed with 1X Passive Lysis Buffer to assay luciferase activity. Parallel protein lysates were prepared with whole-cell extract lysis buffer for Western blotting analysis using Anti-Luciferase pAb for firefly luciferase and an anti-FLAG antibody for NanoLuc™ luciferase.

Five-day-old CD-1 mice were infected with 1,000 PFU of SINV reporter viral stock in the ventral thorax region while six to eight-week-old CD-1 mice were infected with 1,000 PFU of EEEV reporter viral stock in the right rear footpad, and monitored for at least ten days. Groups of mice were sacrificed at various intervals, tissues (e.g., brain and spleen) homogenized in 1X Passive Lysis Buffer, frozen at –80°C and luciferase activity analyzed. For imaging studies, adult C57Bl/6 IFNAR1-/- mice were infected in both hind limb footpads with 1,000 PFU of either firefly or NanoLuc™ luciferase-TaV viral stocks. Six, 24 or 48 hours post-infection, 3mg of D-luciferin or 10μg of furimazine were injected into the tail vein. Mice were imaged for 2 seconds within 2 minutes of substrate administration. (4442)

Notes: This paper describes a bioluminescence-based method for the detection of DNA methyltransferase activity based on methylation-resistant cleavage and protein expression. The authors used Dam methylase as a model enzyme, MboI as the methylation-resistant endonuclease, and luciferase reporter DNA (LR-DNA) as the target. LR-DNA was amplified by PCR and then used as substrate in a Dam methylation reaction. If the target sites in the LR-DNA were fully methylated, they were resistant to subsequent MboI cleavage and expressed luciferase upon in vitro transcription/translation. Incomplete methylation or the absence of methylation resulted in DNA digestion and diminished/absent luciferase activity. TNT® T7 Quick for PCR DNA was used for in vitro translation of the LR-DNA, and the Luciferase Assay System and GloMax® 20/20 Luminometer were used to measure luciferase activity. (4191)

Notes: These authors developed a protein-aggregation reporter that uses huntingtin exon 1 containing 72 glutamines fused to the N-terminal end of firefly luciferase (httQ72-Luc). This construct did not aggregate unless seeded by a non-luciferase-containing polyglutamine (polyQ) protein. Upon co-aggregation, httQ72-luc becomes insoluble and loses its enzymatic activity. httQ72-Luc and a polyQ protein were used to screen a library for compounds that prevent polyQ aggregation. Leflunomide and its active metabolite teriflunomide inhibited protein aggregation independently of their known role in pyrimidine biosynthesis. This study demonstrated the usefulness of luciferase-based protein aggregate reporters for high-throughput screening applications. Luciferase activity was measured using the Luciferase Assay System and the GloMax®-Multi Detection System. (4188)

Notes: These authors investigated the effects of various anesthetics on bioluminescence imaging with firefly luciferase. They observed decreases in luminescence with volatile anaethetics, and found increased luciferase expression with injectable anaethetics in intact cells, but not in cell lysates in vitro. They concluded that the decreases in luciferase activity observed with volatile anaesthesia were due to hemodynamic effects, and not due to a direct inhibitory effect on luciferase enzyme itself. The apparent enhancement of luciferase activity with certain injectable anaesthetics appeared to be due to cytotoxic effects that resulted in increased permeablity to luciferase, as the same enhancement was not observed in cell lysates. D-Luciferin was used for in vivo imaging experiments. The pGL4.10 vector (encoding firefly luciferase), Luciferase Assay Reagent, and the GloMax® 96 Microplate Luminometer were used for in vitro assays using cell lysates. (4190)

Notes: The authors of this paper investigated the molecular mechanisms through which fumaric acid esters affect the progression and pathology of multiple sclerosis. The authors looked at the activation of the Nrf2 transcriptional pathway which plays a role in defense against oxidative stress. A stable reporter cell line expressing eight copies of the gluthione-S-transferase 2 antioxidative response elements cloned upstream of the luciferase complementary DNA in a pGL4.26 vector. Reporter cells were stimulated with dimethylfumarate for 24 hours and luciferase activity measured using the Luciferase Assay System. A strong dose-dependent induction of antioxidative response elements was observed. Next, the authors looked at the effects of fumaric acid esters on the inhibitor of Nrf2, Keap 1. C-terminal, V5-tagged Keap1 protein was transfected into 293 cells. Forty-eight hours post transfection, cells were treated with dimethyl fumarate. Samples were lysed, Keap1-V5 was immunoprecipitated and gel purified. The protein was digested with trypsin in the presence of ProteaseMAX™ Surfactant, Trypsin Enhancer. Peptide pools were analyzed using liquid chromatography-tandem mass spectrometry. The authors were able to show that the Keap1 protein was modified in response to treatment. (4215)

Notes: Human tumor cell lines were subjected to therapeutic doses of ionizing radiation, and MET mRNA, and protein expression and signal transduction were compared in treated and untreated cells. Luciferase reporter assays were used to evaluate the activity of the MET promoter in irradiated cells. The construct pGL2-3.1 encoding the luciferase gene under control of the human full-length MET promoter and a control promoterless pGL2-Basic Vector were transfected into cells prior to irradiation. For RNA interference experiments, cells were transfected with siRNAs 24 hours before the promoter plasmids were introduced. Luciferase activity was measured on a GloMax® 96-well Microplate Luminometer using the Luciferase Reporter Assay System. After irradiation, MET expression in cell lines was increased up to fivefold. (4200)

Notes: The authors of this review article highlight the use of bioluminescence as a readout for high-throughput ADME/Tox assays. They discuss three strategies for designing bioluminescent assays, using either luciferase, ATP or luciferin substrates as the limiting reagents for a luciferase-catalyzed reaction. Reporter gene assays limit the production of luciferase by tying it to a promoter or DNA regulatory region of interest. Such assays can be used to study genes that are regulated by drugs and other xenobiotics. Bioluminescent assays in which ATP is the limiting reagent of the luciferase reaction can be designed to monitor cell viability or the activity kinases. Bioluminescent assays in which the substrate is limiting can be designed so that the activity of a particular enzyme results in the production of a luciferin substrate that can, in turn, be acted upon by luciferase. (3926)

Notes: This study investigated the mechanism of silencing of the estrogen receptor α mRNA in the human breast cancer cell line, MCF-7. The authors initially used software for miRNA target prediction to analyze the 3´ UTR of the human ERα gene for potential miR-206 target sites. Two potential targets, designated hERα1 and hERα2, were identified. ERα levels were repressed in a dose-dependent manner in MCF-7 cells transfected with a synthetic pre-miR-206 duplex, and transfection of an miR-206 expression construct into MCF-7 cells also resulted in specific inhibition of ERα expression, as measured by real-time PCR and Northern blot assays. A luciferase reporter assay was then used to determine whether miR-206 interacted directly with the hERα1 and hERα2 sites in the ERα 3´UTR. Luciferase reporter constructs containing either the hERα1 or hERα2 cloned 3´ of the firefly luciferase gene showed miR-206-medisted repression of luciferase expression in HeLa cells. Mutation of the hERα1 or hERα2 sites to disrupt hybridization with the 5´ region of miR-206 restored luciferase activity, as did co-transfection with an miRNA antagonist of miR-206. Transformation of the luciferase constructs into the breast cancer cell lines, MCF-7 and MDA-MB-231, both of which expressed high levels of miR-206 as measured by real-time PCR, resulted in repression of luciferase activity. Treatment with estrogen was then shown to reduce miR-206 levels in MCF-7 cells. Luciferase assays were used to confirm this result, and levels of luciferase activity from the reporter constructs were increased upon exposure to estrogen, indicating that ERα agonists were able to decrease miR-206 levels in MCF- cells. (3603)

Notes: TSP50 is a testis-specific gene found to be overexpressed in human breast cancer tissue. Of interest is a putative p53 binding site in the TSP50 promoter. To examine what effect p53 may have on TSP50 expression, the TSP50 promoter was cloned into a pGL3 Luciferase Reporter Vector and cotransfected with a wildtype or R249S mutant p53 and a control vector, pCMV/β-galactosidase, into HeLa, HEK293 and paired MCF7 cells. After 24 hours, the cells were assessed for luciferase expression using the Luciferase Assay System and normalized to β-galactosidase expression, which was measured using the Beta-Glo® Assay System. (3598)

Notes: To examine the putative binding of the poliovirus nonstructural proteins to each other, the wildtype and mutant 2B, 2C, 2BC, 3A, and 3AB regions were amplified and cloned into the CheckMate™ Mammalian Two-Hybrid System vectors, pACT and pBIND, after digestion with Sal I and Mlu I. COS cells grown in 12-well plates were transfected with a total of 1.6µg DNA. To reduce competition for transcription factors, the mix of pBIND:pACT:pG5luc plasmids was adjusted from 1:1:1 to 0.053µg pBIND, 0.053µg pACT with a higher concentration of pG5luc (1µg) and an additional 0.49µg of carrier pGEM®-3. After 24 to 28 hours, the firefly luciferase activity was assessed using 10µl of cell lysate with 100µl Luciferase Assay Reagent and measuring the luminescence. (3492)

Notes: Having observed that blunt 27mers had increased potency in RNAi compared to 21mers or 27mers with 3' or 5' overhangs, these authors investigated what differences may account for these changes in gene silencing activity using the same target sequence in enhanced green fluorescent protein (EGFP). For one experiment, a PCR-generated fragment of the EGFP coding region spanning sites EGFPS1 and EGFPS2 was cloned into psiCHECK™-2 Vector in both sense and antisense orientations. Also, a PCR-generated fragment of the human heterogeneous nuclear ribonucleoprotein H (hnRNPH) coding region spanning sites H1 and H3 was similarly cloned in sense and antisense orientations. HEK293 cells were transfected with 150ng EGFP sense and antisense vectors plus EGFPS2 or control duplex RNAs. HCT116 cells were transfected with 100ng sense and antisense hnRNPH vectors with H3 or control duplex RNAs. The Dual-Luciferase® assay was used to evaluate luciferase expression 24 hours post-transfection. In a separate EGFP RNAi experiment, the Steady-Glo® Luciferase Assay System was used to monitor firefly luciferase activity to normalize transfection of HEK 293 cells.
A further RNAi experiment targeted the firefly luciferase gene in the pGL3-Control Vector cotransfected with 20, 2 or 0.4 nM siRNA duplexes into HeLa cells. After 48 hours, the cells were lysed and 10µl tested using the Luciferase Assay System. To test the level of expression of human La antigen targeted for gene silencing, total RNA was harvested from HeLa cells using the SV 96 Total RNA Isolation System, reverse transcribed and used in real-time PCR. (3289)

Notes: Wildtype or p53-knockout mouse embryo fibroblasts (MEFs) were infected with 2 MOI A/Puerto Rico/8/34 (PR8) influenza virus. After washing, DMEM containing 1% FBS was added and the cells were grown for 8 or 24 hours before adding the Caspase-Glo® 3/7 Reagent. After 1 hour, luminescence was measured and the fold increase was determined by comparing the RLUs of infected with mock-infected cells. In addition, the Luciferase Assay System was used to assess the level of p53 induction after PR8 infection. Either human primary lung or A549 cells were transfected with a p53-responsive firefly luciferase vector, subsequently infected with 2 MOI PR8 and assessed for reporter enzyme activity 4–24 hours post-infection. (3318)

Notes: A fragment containing HIV-1 LAI 5' LTR was cloned into the unique EcoICRI-XhoI site of the pGL3-Basic Reporter Vector. The Luciferase Reporter Assay was used to analyze DNA transfected cells for luciferase activity. The pRL-TK Vector was used as a transfection efficiency internal control with a Renilla cDNA under control of HSV-TK. Firefly luciferase activity derived from the HIV-1 LTR was normalized to the Renilla luciferase activities using the Dual-Luciferase® Reporter Assay. (3703)

Notes: In this paper, the effect of a 5’ untranslated region from the Bcl-2 gene transcripts on firefly and Renilla reporter constructs was evaluated. A number of studies were performed using various single- and dual-reporter constructs containing the Bcl-2 5’ UTR. These constructs were transfected into 293T cells and assayed for luciferase activity using the Dual-Luciferase® Reporter Assay System. Transfection studies with firefly luciferase mRNA constructs were also performed. In these experiments, firefly luciferase levels were measured using the Luciferase Assay System. Transfections were normalized using the pSV-β-Galactosidase Control Vector and the Beta-Glo® Assay System. (3125)

Notes: Leaves of Glycine max (soybean) were co-transfected by particle bombardment with various combinations of vectors encoding plant disease resistance (R) genes and a luciferase reporter construct containing the constitutive 35S promoter of Cauliflower mosaic virus. Leaf disks from the transfected areas were frozen in liquid nitrogen, ground, and resuspended in 240μl of Cell Culture Lysis Reagent. The lysates were then assayed for luciferase activity with the Luciferase Assay System. The luciferase values correlated to plant leaf cell survival of the various constructs. (3035)

Notes: Human polynucleotide phosphorylase (hPNPaseold-35) was originally identified as a gene that is upregulated during cellular differentiation and senescence. The authors of this study show that overexpression of hPNPaseold-35 results in the increased production reactive oxygen species (ROS) and the activation of the NF-κB pathway, resulting in expression of the inflammatory cytokines IL-6 and IL-8. HeLa cells were transfected with either empty pGL3-Basic or 3kB-Luc (pGL3-Basic containing three tandem NF-κB binding sites). Transfected cells were infected with an empty adenoviral vector or one containing hPNPaseold-35. Luciferase assays were conducted using the Luciferase Assay System, and signal was normalized to a pSV-βgal control. A 10- to 12-fold increase in luciferase activity was observed in the cells infected with the adenovirus containing hPNPaseold-35 compared to the control cells.This activation was inhibited by compounds that reduced ROS. The authors suggest that hPNPaseold-35 plays an important role in producing age-related inflammation. (3643)

Notes: In this article, a 2.5- and a 1.7-kb fragment of the myostatin promoter were amplified by PCR and cloned into the pGEM®-T Easy vector. These clones were then subcloned into the pGL3-Basic Vector. Deletion mutants of the constructs were made and used to transient transfect mouse muscle C2C12 cells. The Luciferase Assay System was then used to analyze transfectants. The researchers also injected luciferase-reporter constructs into mouse muscle. Luciferase activity in the mouse muscle was examined by grinding isolated muscles in liquid nitrogen and resuspending them in Cell Culture Lysis Buffer. Ten micron cryosections of the muscles were also used in immunohistochmical staining experiments. For these experiments, the researchers used a 1:50 dilution of Promega’s polyclonal anti-luciferase antibody, a secondary antibody and tertiary fluorescein-labeled conjugate. The slides were counter stained with DAPI. (3147)

Notes: The Beta-Glo® Assay System was used to normalize luciferase readings in dual-reporter transfection studies. In these experiments, human prostate carcinoma PC-3 cells were transfected with a NF-κB factor-driven luciferase reporter construct and a undefined beta-galactosidase reporter construct. Cells were treated with 20 and 40µM apigenin for 16 h or with 10ng/ml TNF-α for 30 minutes. Luciferase reporter enzyme expression was assessed with the Luciferase Assay System. (3054)

Notes: To test the effect of BMS-378806, a new small molecule inhibitor of HIV-1, a cell fusion assay was developed. Target cells that stably expressed CD4, CXCR4 or CCR5 receptors and carry a responsive luciferase plasmid were prepared. Effector cells were transiently transfected with HIV coat protein gp160 from various strains of virus, and a plasmid to activate the responsive element promoting luciferase. Therefore, if the cells fuse, luciferase is activated. To measure this activation, effector cells were plated with target cells in a 1:2 ratio and seeded into 96-well plates at 1.5 x 104 cells/well and incubated with various concentrations of BMS-378806 for 12-24 hours. Luciferase activity was determined with the Steady-Glo® Reagent. (2737)

Notes: Total RNA was isolated from human testes using the RNAgents® Total RNA Isolation System. The isolated RNA was used in reverse transcription reactions to make cDNAs of HLA-linked OR genes. Promoter regions of HLA-linked OR genes were cloned into the pGL3-Basic Vector. The constructs were then transfected into human embryonic kidney (HEK293) and Odora cells for promoter analysis studies. The Bright-Glo™ Luciferase Assay System was used to generate data from the study. Results were presented as a comparison to pGL3-Control Vector transfectants. (2730)

FEBS Lett.555, 390-396.
Hsp105 but not Hsp70 family proteins suppress the aggregation of heat-denatured protein in the presence of ADP.2003

Yamagishi, N., Ishihara, K., Saito, Y., and Hatayama, T.

Notes: The pGL3-Control Vector was co-transfected into COS-7 cells with mammalian expression vectors expressing either heat shock protein Hsp70 or Hsp105α. The cells were ATP-depleted in glucose-free media with 2μM rotenone and 5mM 2-deoxyglucose for up to 3 hours. Afterwards the cells were lysed with the Cell Culture Lysis Reagent and assayed for luciferase activity uaing the Luciferase Assay System. ATP depletion was verified during 2μM rotenone and 5mM 2-deoxyglucose treatment using the CellTiter-Glo® Assay. (2841)

Notes: Researchers used Promega's pGL3-Basic Vector to create a NF-κB promoter-driven firefly luciferase reporter vector for use in transient transfections with the phRL-TK Vector. The two vectors, along with pUC18, were transiently transfected into HEK293 cells using GeneJuice Transfection Reagent (Novagen). Transfections were performed oversnight using 80ng NF-κB-pGL3-Basic construct, and 20ng of phRL-TK and pUC18 vectors in 96-well plates containing 2.5 x 104 cells/well. The following day, the cells were exposed to various treatments. Luciferase activity was measured using the Dual-Glo™ Luciferase Assay System. (2672)

J. Virol.77, 1992-2002.
Molecular and functional analysis of an interferon gene from the zebrafish, Danio rerio.2003

Altmann, S.M., Mellon, M.T., Distel, D.L. and Kim, C.H.

Notes: The pGEM®-T Easy Vector was used to subclone products of a 5´ RACE reaction. A promoter construct, assembled in the pGL3 Basic Vector, was co-transfected with a zebrafish interferon expression vector in the ZF4 zebrafish embryo fibroblast cell line using the TransFast™ Reagent (details provided). Luciferase levels were examined with the BrightGlo™ Luciferase Assay Reagent. Induction of zebrafish mRNA was also examined in zebrafish liver cells (ZFL) following treatment with the known interferon inducer, poly(I)-poly(C). RNA was extracted and reverse transcribed using ImProm-II™ Reverse Transcriptase. The resulting cDNA was used for quantitative, real-time RT-PCR with a SYBR green-based assay. (2627)

Notes: The pGL3-Promoter Vector was treated with various concentrations of the chemotherapeutic cancer and DNA cross-linking agent, cisplatin. The damaged pGL3-Promoter Vector and the pSV-β-Galactosidase Control Vector were then transiently transfected into the human lung adenocarcinoma cell line, A549, and later analyzed for luciferase activity using the Luciferase Assay System with Reporter Lysis Buffer. The researchers also used M-MLV Reverse Transcriptase to clone the antisense sequence to XPA (Xeroderma Pigmentosum group A) mRNA. The cloned antisense sequence was then expressed in A549 cells for its effect on XPA gene expression. (2833)

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